CRAF (IC50 = 0.072 nM); Braf (IC50 = 0.21 nM); ARAF (IC50 = 6.4 nM); p38α (IC50 = 2.1 μM); Abl1 (IC50 = 4.9 μM)

LXH254 (Compound A) is an adenosine triphosphate (ATP)-competitive inhibitor of BRAF (also referred to herein as b-RAF or b-Raf) and CRAF (also referred to herein as c-RAF or c- Raf) protein kinases. LXH254 is also referred to as a C-RAF/c-Raf kinase inhibitor and as a c-RAF (or CRAF) inhibitor throughout the present disclosure. In cell-based assays, LXH254 has proven to have anti-proliferative effects in cell lines with a variety of mutations that stimulate MAPK signaling. Additionally, LXH254 is a Type 2 ATP-competitive inhibitor of both B-Raf and C-Raf that maintains the kinase pocket in an inactive conformation, reducing the paradoxical activation seen with many B-Raf inhibitors and inhibiting mutant RAS-driven signaling and cell proliferation[1].
LXH254 (0–10 µM, 1 h) inhibits monomeric and dimeric RAF and encourages the formation of RAF dimers[2].
The ability of LXH254 to inhibit MAPK signaling driven by ARAF is decreased, and it has also been shown that when CRAF expression is absent, ARAF's contribution to MAPK signaling increases[2].
LXH254 shows more sensitivity when cells lack ARAF[2].

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Researchers describe an unexpected paralog selectivity of Naporafenib (LXH-254), which is able to potently inhibit BRAF and CRAF, but has less activity against ARAF. LXH254 was active in models harboring BRAF alterations, including atypical BRAF alterations coexpressed with mutant K/NRAS, and NRAS mutants, but had only modest activity in KRAS mutants. In RAS-mutant lines, loss of ARAF, but not BRAF or CRAF, sensitized cells to LXH254. ARAF-mediated resistance to LXH254 required both kinase function and dimerization. Higher concentrations of LXH254 were required to inhibit signaling in RAS-mutant cells expressing only ARAF relative to BRAF or CRAF. Moreover, specifically in cells expressing only ARAF, LXH254 caused paradoxical activation of MAPK signaling in a manner similar to dabrafenib.

In a number of KRAS-mutant models, including the NSCLC-derived Calu-6 (KRAS Q61K) and NCI-H358 (KRAS G12C), treatment with LXH254 (Compound A) results in tumor regression. Numerous MAPK-driven human cancer cell lines and xenograft tumors that represent model tumors with human lesions in the KRAS, NRAS, and BRAF oncogenes show efficacy for LXH254[1].
In models with BRAF mutations, whether they are present alone or in combination with activated NRAS or KRAS, LXH254 exhibits significant antitumor activity, and RAS mutants deficient in ARAF are more responsive to LXH254[2].

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RAS mutants lacking ARAF are more sensitive to Naporafenib (LXH-254) in vivo [2]
We next determined the effect of ARAF ablation on sensitivity to MAPK inhibitors in vivo. Tumors formed from parental and ARAF-deleted variants of the HCT 116, MIA PaCa-2, and MEL-JUSO models were treated with vehicle, 0.3 mg/kg every day trametinib, or 50 mg/kg twice a day Naporafenib (LXH-254). Doses of trametinib and LXH254 were selected that matched AUC values for the approved dose of trametinib (2 mg, twice a day) or the recommended phase II dose of LXH254 (400 mg, twice a day). ARAF-deleted xenografts either grew with similar kinetics (HCT 116 cells) or slightly slower (MIA PaCa-2 and MEL-JUSO) than their parental counterparts, indicating that ARAF ablation has at most modest effects on the fitness of these models. In all parental models, LXH254 and trametinib exerted similar effects on tumor growth, resulting in a slow growth phenotype, which was most pronounced in MEL-JUSO cells (Fig. 7A–C). Treatment with LXH254 in the ARAF knockouts led to complete regression of HCT 116 and MEL-JUSO and near-complete regression of MIA PaCa-2 xenografts. Several tumors from each KRAS-mutant models regrew after prolonged treatment, including three of six from the HCT 116 and five of six of the MIA PaCa-2 tumors (Supplementary Fig. S4A–S4C). Initial analysis of these variants by Western blot analysis suggested that at least two tumors (MIA PaCa-2, M2, and M5) became resistant due to RAF pathway reactivation, with reactivation in one case potentially attributable to restoration of ARAF expression (M5). Lack of clear pathway reactivation in the other six tumors suggested resistance occurred via a MAPK bypass mechanism (Supplementary Fig. S4D). Strikingly, two of three of the regressed HCT 116 tumors failed to regrow after cessation of drug treatment consistent with complete eradication of the tumor (Supplementary Fig. S4A). In contrast, variants lacking ARAF expression had similar sensitivities to trametinib as parental MIA PaCa-2 cells.
These data indicate that RAS-driven activation of BRAF and CRAF is inhibited sufficiently by Naporafenib (LXH-254) to eradicate tumors, and conversely that poor ARAF inhibition is a critical contributor to the relative insensitivity of RAS-mutant models to LXH254. Accordingly, we tested whether cotreatment of tumors with LXH254 and low doses of trametinib, aimed at inhibiting residual ARAF-driven signaling, improved antitumor effects in MIA PaC-2 cells. Combining LXH254 with either 50% (0.3 mg/kg, every 2 days) or 10% (0.03 mg/kg once a day) doses of trametinib improved efficacy relative to both LXH254 and a full dose of trametinib (0.3 mg/kg, twice a day), although efficacy was inferior to that seen for single-agent LXH254 in cells lacking ARAF (Fig. 7D), and tumors regrew over time (data not shown). Thus, combining LXH254 with even small amounts of an MEK inhibitor can result in significant antitumor effects.

RAF in vitro enzyme assays [2]
Biochemical inhibition of ARAF, BRAF, and CRAF, shown in Fig. 1A, was performed as described for RAF709 in Shao and colleagues. In brief, the catalytically inactive MEK1K97R variant was used as a substrate for either full-length BRAF, full-length activated CRAF (Y340E/Y341E variant), or a full-length N-terminal GST-ARAF fusion. In all cases, Naporafenib (LXH-254) was preincubated with CRAF/BRAF/ARAF for 30 minutes prior to substrate addition/reaction initiation. Biochemical activity of Naporafenib (LXH-254) in the extended panel of kinases, shown in Supplementary Table S1, was determined as described in Wylie and colleagues.

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Structural modeling [2]
The in-house X-ray structure (PDB entry: 6N0P) of Naporafenib (LXH-254) bound to BRAF was used in conjunction with an X-ray structure of CRAF (PDB entry: 3OMV) to build a homology model of ARAF with Naporafenib (LXH-254) bound in the molecular operating environment. This model was extensively refined with explicated solvent molecular dynamics simulations at 300 K, 1 atm, AM1BCC/ELF charges, TIP3P water, and ff14SB force field with the PARM@FROSST extension within the AMBER suite of simulation tools. Ten-nanosecond simulations for each system were run and time average structures were extracted.

Cell-based kinase assays [2]
In-cell kinase selectivity profiling was performed by KiNativ. Briefly, HCT 116 cells were treated with Naporafenib (LXH-254) for 2 hours at 10 μmol/L, and then lysates were processed, probe labeled, and analyzed by LC/MS-MS, as described previously. For immunoprecipitate (IP)-kinase assays, cells were cultured in compound for 4–24 hours prior to protein immunoprecipitation, as described above. IPs were washed once with IP lysis buffer and twice with 1 × Kinase Buffer, and protein-bound beads were incubated in 2 × kinase buffer with 250 μmol/L ATP and 0.5 μg MEK1 or MEK1-K97M for 30 minutes at 30°C. Beads were then washed three times with IP lysis buffer and prepared for immunoblotting as described above.

Outbred athymic (nu/nu) female mice and SCID Beige mice; BRAF-, NRAS-, and KRAS-mutant xenograft models, as well as a RAS/RAF wild-type model[2]
100 mg/kg
Orally, daily

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Cell culture and in vivo efficacy [2]
All cell lines were shown to be free of Mycoplasma species and murine viruses in the IMPACT VIII PCR Assay Panel (IDEXX RADIL, IDEXX Laboratories Inc). In all cases, cells were harvested at 80%–95% confluence with 0.25% trypsin-EDTA, washed with PBS, detached with 0.25% trypsin-EDTA, and neutralized with growth medium. Following centrifugation for 5 minutes at 1,200 rpm, all cells, except HEY-A8 cells (PBS), were resuspended in either cold Hank's Balanced Salt Solution (HBSS; HCT 116 and A375) or cold HBSS and an equal volume of Matrigel Matrix. Cells were then injected in 100–200 μL volumes into either the right or left flanks of female nude mice, with the exception of MEL-JUSO cells, which were injected into SCID beige mice. Total cell numbers injected per mouse were 2 × 106 (HCT 116 and HEY-A8), 5 × 106 (MIA PaCa-2, HPAF-II, A375, Hs944.T, and SK-MEL-30), or 1 × 107 (MEL JUSO, Calu-6, and PC-3). Five to 7 mice were used for each group.
3990HPAX, 2043HPAX, 1290HCOX, 2861HCOX, 1855HCOX, 2094HLUX, and 3486HMEX patient-derived tumor xenograft tumors and the WM793 xenograft were propagated by serial passage of tumor fragments in nude mice. Briefly, 3 × 3 × 3 mm fragments of fresh tumor from a previous passage were implanted subcutaneously into the mice. Efficacy was carried with n = 6–12 mice per group.
Percent change in body weight (BW) was calculated as (BWcurrent − BWinitial)/(BWinitial) × 100%. Data were presented as mean percent body weight change from the day of treatment initiation ± SEM. Tumor volume was determined as published previously. All data were expressed as mean ± SEM. Changes in tumor volume were used for statistical analysis, and between-group comparisons were carried out using a one-way ANOVA. For all statistical evaluations, the level of significance was set at P < 0.05. Significance compared with the vehicle control group is reported, unless otherwise stated.

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Drug formulation [2]
Naporafenib (LXH-254) was dosed orally in MEPC4 vehicle [45% Cremophor RH40 + 27% PEG400 + 18% Corn Oil Glycerides (Maisine CC) + 10% ethanol] for all experiments, except PC3, where Naporafenib (LXH-254) was formulated in 90% PEG400 + 10% Tween80. MEPC4 stock was diluted 5 × (1:4 with de-ionized water) prior to dosing. Trametinib was dosed orally as a suspension in 0.5% HPMC and 0.2% Tween80 in distilled water at pH 8. Trametinib was stirred overnight and protected from light at room temperature prior to dosing.

Tolerability and safety
Study treatment was discontinued in all patients. The primary reason was progressive disease (PD) (Supplementary Table 3).
In naporafenib escalation and naporafenib/spartalizumab escalation and expansion, at least one naporafenib dose reduction was reported in 14 (16.1%; due to AEs: 3 [3.4%]), 6 (50.0%; due to AEs: 5 [41.7%]) and 20 (46.5%; due to AEs: 10 [23.3%]) patients, respectively, and at least one naporafenib dose interruption was reported in 87 (100%; due to AEs: 34 [39.1%]), 12 (100%; due to AEs: 9 [75.0%]) and 42 (97.7%; due to AEs: 20 [46.5%]) patients, respectively.
In the single-agent arm, five of 68 (7.4%) patients experienced at least one DLT: grade 4 decreased platelet count (one patient; 1200 mg QD), grade 3 neuralgia and grade 3 maculopapular rash/grade 3 pruritus (two patients; 600 mg BID), and grade 3 blood bilirubin increased/grade 3 hyponatremia and grade 3 peripheral sensory neuropathy (two patients; 800 mg BID). No MTD was formally declared for single-agent naporafenib at the highest tested dose of 1200 mg in the QD regimen. The MTD/RDE was formally determined to be 600 mg for the singleagent naporafenib BID regimen. Given that both the QD and BID dosing regimens were expected to have the same safety profile depending on the relative dose intensity, the BID regimen was chosen over the QD regimen to reduce pill burden. The combination dose-escalation arm started at one dose level below the MTD (400 mg BID). No DLTs were reported after treatment with naporafenib 400 mg BID/spartalizumab or naporafenib 600 mg BID/spartalizumab. The MTD for naporafenib/spartalizumab was formally not reached. Due to safety concerns (grade 3 rash) of naporafenib 600 mg BID/spartalizumab, the RDE for naporafenib in the combination was determined to be 400 mg BID.
For the single-agent arm, treatment-related adverse events (TRAEs) of any grade were reported in 79 (90.8%) patients. The most frequent (occurring in ≥ 20% of patients) were dermatitis acneiform (maculopapular pustular eruptions) (21 [24.1%], no grade 3/4 events), rash (21 [24.1%], grade 3/4: 1 [1.1%]), fatigue (18 [20.7%], grade 3/4: 2 [2.3%]) (Table 2, Supplementary Table 4). For naporafenib/spartalizumab escalation, TRAEs of any grade were reported in 11 (91.7%) patients; those that occurred in ≥ 20% of patients were nausea and pruritus (4 [33.3%] each, no grade 3/4 events), and dermatitis acneiform (3 [25%], grade 3/4: 1 [8.3%]) (Table 2, Supplementary Table 5). For dose expansion, TRAEs of any grade were reported in 39 (90.7%) patients. The most frequent TRAE was rash (any-grade: 17 [39.5%], grade 3/4: 6 [14.0%]) (Table 2, Supplementary Table 5). Treatment-emergent AEs reported across different dose levels and regimens are detailed in Supplementary Table 6 and Table 7, and treatment-related SAEs in Supplementary Table 8 and Table 9.
Neurological grade 3 AEs of interest relating to naporafenib and occurring during interruption of single-agent naporafenib treatment or after the end of study treatment were 2 cases of neuralgia and 1 case each of peripheral sensory neuropathy and seizure, while for a patient treated with naporafenib/spartalizumab, was 1 case of myalgia (data on file).
During the on-treatment period in dose escalation, 11 (12.6%) patients in the single-agent arm and one patient and the combination arm died of underlying malignancy. In dose expansion, four (9.3%) patients died (three of underlying malignancy and one due to COVID-19 infection).
https://pmc.ncbi.nlm.nih.gov/articles/PMC11380116/#S12

The median Tmax for naporafenib did not depend on a dose and regimen, and the AUC0-last and Cmax increased in an approximate dose-proportional manner across dose ranges in both schedules (Supplementary Table 10). The median Tmax, Cmax and AUC0-last for naporafenib administered in combination with spartalizumab was 3.08–4.00 h for the 400 mg BID regimen and 2.15 h for the 600 mg BID regimen (Supplementary Table 11). The geometric mean Cmax and AUC0-last were generally consistent with those of single-agent naporafenib at doses of 400 mg BID and 600 mg BID on days 1 and 15 (Supplementary Table 11). The arithmetic mean concentration-time profiles for all arms are shown in Supplementary Figure 2. A statistically significant association between the range of naporafenib Cmax and the probability of developing grade ≥ 2 rash was observed (Supplementary Figure 3). Assessment of rBA and immunogenicity results are described in Supplementary Data. https://pmc.ncbi.nlm.nih.gov/articles/PMC11380116/#S12

[1]. THERAPEUTIC COMBINATIONS COMPRISING A RAF INHIBITOR AND A ERK INHIBITOR. WO 2018051306 A1 20180322

[2]. LXH254, a Potent and Selective ARAF-Sparing Inhibitor of BRAF and CRAF for the Treatment of MAPK-Driven Tumors. Clin Cancer Res. 2021 Apr 1;27(7):2061-2073.

Naporafenib is an orally available inhibitor of all members of the serine/threonine protein kinase Raf family, with potential antineoplastic activity. Upon administration, naporafenib binds to Raf proteins and inhibits Raf-mediated signal transduction pathways. This inhibits proliferation of Raf-overexpressing tumor cells. Raf protein kinases are critical enzymes in the Ras/Raf/MEK/ERK signaling pathway and are upregulated in a variety of cancer cell types. They play key roles in tumor cell proliferation and survival.

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Purpose: Targeting RAF for antitumor therapy in RAS-mutant tumors holds promise. Herein, we describe in detail novel properties of the type II RAF inhibitor, Naporafenib (LXH-254). Experimental design: Naporafenib (LXH-254) was profiled in biochemical, in vitro, and in vivo assays, including examining the activities of the drug in a large panel of cancer-derived cell lines and a comprehensive set of in vivo models. In addition, activity of Naporafenib (LXH-254) was assessed in cells where different sets of RAF paralogs were ablated, or that expressed kinase-impaired and dimer-deficient variants of ARAF.
Results: We describe an unexpected paralog selectivity of Naporafenib (LXH-254), which is able to potently inhibit BRAF and CRAF, but has less activity against ARAF. Naporafenib (LXH-254) was active in models harboring BRAF alterations, including atypical BRAF alterations coexpressed with mutant K/NRAS, and NRAS mutants, but had only modest activity in KRAS mutants. In RAS-mutant lines, loss of ARAF, but not BRAF or CRAF, sensitized cells to LXH254. ARAF-mediated resistance to LXH254 required both kinase function and dimerization. Higher concentrations of LXH254 were required to inhibit signaling in RAS-mutant cells expressing only ARAF relative to BRAF or CRAF. Moreover, specifically in cells expressing only ARAF, LXH254 caused paradoxical activation of MAPK signaling in a manner similar to dabrafenib. Finally, in vivo, LXH254 drove complete regressions of isogenic variants of RAS-mutant cells lacking ARAF expression, while parental lines were only modestly sensitive.
Conclusions: LXH254 is a novel RAF inhibitor, which is able to inhibit dimerized BRAF and CRAF, as well as monomeric BRAF, while largely sparing ARAF. [2]

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Consistent with this hypothesis, we found that adding even low doses of trametinib to a full dose of LXH254 improved antitumor activity relative to single-agent effects in the MIA PaCa-2 model (Fig. 7D). The observation that adult mice tolerate simultaneous loss of BRAF and CRAF (8) suggests that poor ARAF inhibition may improve LXH254 tolerability relative to, for example, MEK and ERK inhibitors, thereby facilitating the achievement of drug exposures where both BRAF and CRAF are strongly inhibited. Such improved tolerability might also facilitate combinations with additional MAPK inhibitors for the treatment of RAS-mutant tumors, resulting in improved pathway suppression and antitumor effects. In support of this idea, LXH254-anchored MAPK combinations, such as those described in Fig. 7, display a superior tolerability/efficacy relationship when compared with single-agent treatments in preclinical species (manuscript in preparation). Moreover, combinations between LXH254 and the MEK inhibitor, trametinib, and ERK inhibitor, LTT462, are currently in dose expansion in patients with a variety of RAS/RAF pathway alterations.

C25H25F3N4O4

502.4856

502.49

C, 59.76; H, 5.01; F, 11.34; N, 11.15; O, 12.74

1800398-38-2

1800398-38-2;LXH254 HCl;

90456533

Off-white to light yellow solid powder

3.2

2

10

7

36

709

0

FC(C1C=C(C=CN=1)C(NC1C=CC(C)=C(C=1)C1C=C(N=C(C=1)N1CCOCC1)OCCO)=O)(F)F

UEPXBTCUIIGYCY-UHFFFAOYSA-N

InChI=1S/C25H25F3N4O4/c1-16-2-3-19(30-24(34)17-4-5-29-21(12-17)25(26,27)28)15-20(16)18-13-22(32-6-9-35-10-7-32)31-23(14-18)36-11-8-33/h2-5,12-15,33H,6-11H2,1H3,(H,30,34)

N-[3-[2-(2-hydroxyethoxy)-6-morpholin-4-ylpyridin-4-yl]-4-methylphenyl]-2-(trifluoromethyl)pyridine-4-carboxamide

LXH-254; Naporafenib; LXH254; LXH254; Naporafenib; 1800398-38-2; Naporafenib [INN]; N-(3-(2-(2-hydroxyethoxy)-6-morpholinopyridin-4-yl)-4-methylphenyl)-2-(trifluoromethyl)isonicotinamide; LXH254 free base; Pan-raf inhibitor LXH254; LXH 254

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~100 mg/mL (~199.0 mM)
Ethanol: ~100 mg/mL (~199.0 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.98 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (4.98 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (4.98 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 2.5 mg/mL (4.98 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)